The aim of this study is the elucidation of the mechanisms involved in the transport of vitamin B12 across the cell envelope of Escherichia coli. This process requires the products of the genes BtuB, BtuC, and TonB. The BtuB gene product is an outer membrane protein which binds B12 tightly and the release of B12 from this receptor, probably directly into the periplasmic space, is an energy-dependent process that requires the proton motive force and the TonB gene product. Passage of B12 across the inner membrane requires the BtuC gene product and may also require the input of phosphate bond energy. We propose to examine the interactions between the receptor, the TonB gene product and the proton motive force that lead to an increased rate of dissociation of the receptor-B12 complex. The energy-dependence and other requirements of B12 transport across the inner membrane will also be studied. We have prepared 3H-labeled derivatives of vitamin B12 for use as photo-affinity labels to assist in the identification of those envelope components that participate in B12 transport.